Summary

Mammalian basic helix-loop-helix proteins of the achaete-scute
family are proneural factors that, in addition to the central nervous system,
are required for the differentiation of peripheral neurons and sensory cells,
derivatives of the neural crest and placodal ectoderm. Here, in identifying
the molecular nature of the pia mutation, we investigate the role of
the zebrafish achaete-scute homologue ascl1a during
development of the adenohypophysis, an endocrine derivative of the placodal
ectoderm. Similar to mutants deficient in Fgf3 signaling from the adjacent
ventral diencepahalon, pia mutants display failure of endocrine
differentiation of all adenohypophyseal cell types. Shortly after the failed
first phase of cell differentiation, the adenohypophysis of pia
mutants displays a transient phase of cell death, which affects most, but not
all adenohypophyseal cells. Surviving cells form a smaller pituitary rudiment,
lack expression of specific adenohypophyseal marker genes (pit1,
neurod), while expressing others (lim3, pitx3), and display an
ultrastructure reminiscent of precursor cells. During normal development,
ascl1a is expressed in the adenohypophysis and the adjacent
diencephalon, the source of Fgf3 signals. However, chimera analyses show that
ascl1a is required cell-autonomously in adenohypophyseal cells
themselves. In fgf3 mutants, adenohypophyseal expression of
ascl1a is absent, while implantation of Fgf3-soaked beads into
pia mutants enhances ascl1a, but fails to rescue
pit1 expression. Together, this suggests that Ascl1a might act
downstream of diencephalic Fgf3 signaling to mediate some of the effects of
Fgf3 on the developing adenohypophysis.

INTRODUCTION

The hypothalamic-hypophyseal axis constitutes a functional link between the
nervous and the endocrine system. As a key regulator of hormone secretion, the
hypophysis, also called pituitary gland, controls basic vital processes
including growth, reproduction and overall body homeostasis. The mature
pituitary consists of two functionally and anatomically distinct components:
the neurohypophysis and the adenohypophysis, also named posterior and anterior
pituitary lobe, respectively. In contrast to the neurohypophysis, which
derives from ventral regions of the hypothalamus, the adenohypophysis
originates from placodal ectoderm. At late gastrula and early segmentation
stages, the placodal ectoderm is located at the interphase of the anterior
neuroectoderm and the epidermal ectoderm, constituting a cell population that
in several respects is the neural crest equivalent of the presumptive
forebrain region. Anterior-most placodal ectoderm at the anterior neural ridge
gives rise to the adenohypophysis, while more lateral and posterior regions of
the placodal ectoderm give rise to sensory cells of nose, inner ear and
lateral line, neurons of various cranial ganglia, and the lenses (for review,
see Baker and Bronner-Fraser,
2001).

In contrast to Neurod1, the role of Mash1 in pituitary
development remained elusive. Remarkably, aside from controlling neurogenesis
in the central and peripheral nervous system (CNS, PNS), mouse Mash1 is
crucial for the development of cells of the so-called diffuse neuroendocrine
system, including adrenal medullary chromaffin cells
(Huber et al., 2002), thyroid
parafollicular C-cells (Lanigan et al.,
1998) and pulmonary neuroendocrine cells
(Ball, 2004;
Borges et al., 1997;
Ito et al., 2000). In light of
these data and the common expression of Mash1 in the mammalian
pituitary, it was speculated that Mash1 might play a similar role during the
ontogeny of the adenohypophysis (Ferretti
et al., 2003). However, genetic proof for such a function had been
missing thus far.

Genetic screens in the zebrafish for mutations that disrupt
adenohypophyseal growth hormone (gh) gene expression have
led to the isolation of the pituitary absent (pia) mutant,
characterized by a dearth of lactotrope-, corticotrope-, somatotrope- and
thyrotrope-specific hormone expression during larval stages
(Herzog et al., 2004b). Here,
we report that the pia gene corresponds to zebrafish
achaete-scute-like 1a (ascl1a; formerly called
zash1a) (Allende and Weinberg,
1994), providing genetic evidence for an essential and
cell-autonomous role of this proneural gene during endocrine specification of
adenohypophyseal cells. In the absence of Ascl1a, all adenohypophyseal cell
types fail to express their cognate hormone genes, although only some of them
become apoptotic. This is in contrast to the situation in fgf3
mutants, where failed specification leads to the loss of all adenohypophyseal
cells. In view of this, the ascl1a mutation allows us to distinguish
two different populations of adenohypophyseal cells in which cell
specification and cell maintenance are either coupled or independent from each
other.

MATERIALS AND METHODS

Genetic mapping, identification of mutation and genotyping

The piat25215 mutation was mapped to LG4 via bulked
segregation analysis, using a panel of simple sequence length polymorphism
(SSLP) markers and standard techniques
(Geisler, 2002). Linkage
assignments were confirmed and further refined by genotyping single mutant and
wild-type embryos, which placed piat25215 close to SSLP
marker z27201 (1 recombinant among 192 meiosis). In addition, we identified a
new SSLP, amplifying a genomic fragment from contig BX511171.13 with primers
5′-GTACACTTGAAGCTTGTGCG-3′ and
5′-GTTTTCTGCACCAGAACCTG-3′. For this marker, we found no
recombination among 192 meioses.

The acsl1a open reading frame is encoded by a single exon. To
search for lesions in ascl1a, we amplified overlapping
ascl1a fragments by PCR from genomic mutant and wild-type DNA
samples, followed by sequencing in forward and reverse directions.

The piat25215 mutation generates a HindIII
restriction site, which was used as a restriction fragment length polymorphism
(RFLP) for genotyping. A 523 bp ascl1a fragment containing the
polymorphic restriction site was amplified from genomic DNA of single embryos,
using primers 5′-TCAGAGCATCCAACTCAGCC-3′ and
5′-CGAACGCTCAAAACCAGTTG-3′, followed by HindIII digest.
On wild-type DNA, this results in two fragments of 203 and 320 bp, whereas the
mutant PCR product is cleaved into three fragments of 320, 139 and 64 bp. For
genotyping of pia homozygotes injected with ascl1a BAC DNA,
we identified an RFLP in the 3′-UTR of ascl1a, distinguishing
the wild-type allele from the piat25215 allele and the
BAC-encoded gene. A 387 bp DNA fragment of BAC-injected embryos was amplified
via PCR using primers 5′-AACAAGAGCTCCTGGACTTC-3′and
5′-CACGGTGTCGTGGAAAGTCT-3′, followed by StyI digest,
which cleaved only the piat25215 ascl1a allele and the
BAC-derived fragment (301 bp and 86 bp), but not the endogenous wild-type
allele.

The antisense MO for ascl1a
(5′-CATCTTGGCGGTGATGTCCATTTCG-3′; corresponding to nucleotides -4
to +21 of ascl1a cDNA) was obtained from Gene Tools and diluted in
1× Danieu's buffer to a final concentration of 0.033 pmol/nl. The MO was
injected in volumes of 1 to 1.5 nl into embryos at the one- to four-cell
stage, as described (Nasevicius and Ekker,
2000).

To obtain an ascl1a expression construct, the ascl1a cDNA
was cloned via SmaI/XhoI sites into pCS2+
(Rupp et al., 1994) to yield
pCS2-ascl1a. To test the efficiency of the ascl1a MO (see
below), a fusion construct encoding Ascl1a protein tagged with six C-terminal
Myc epitopes was generated by amplifying the ascl1a-coding region
from pCS2-ascl1a and cloning it into the BamHI and
SpeI sites of pCS2-3′MT (kind gift of U. Strähle). For
ascl1a-VP16 fusion constructs, either the entire
ascl1a-coding region without the termination codon (wild type), or bp
1-207 (mutant) were amplified via PCR, and cloned into the EcoRI site
of pCS2+. The VP-16 activator sequence was excised from a pGMT-VP16 construct
(Pogoda et al., 2000) and
cloned 3′ to the ascl1a fragments. Capped mRNA was prepared
after plasmid linearization, using the Message Machine kit (Ambion). Synthetic
mRNA was injected into one- to four-cell stage embryos, as previously
described (1.5 nl per embryo)
(Hammerschmidt et al., 1999).
For BAC injections, an ascl1a containing BAC was obtained from RZPD
(ID HUKGB735K15264Q8), and injected at a concentration of 75 ng/μl into the
cytoplasm of one-cell stage piat25215 +/- intercross
progeny.

Immunoblotting

Embryos were injected with 800 pg ascl1a-myc mRNA with or without
ascl1a MO, and embryonic protein extraction were collected at 8 hpf
as described (Westerfield,
1994). Protein samples were separated via SDS-PAGE on 12%
Acrylamid/Bis-acrylamid gels, and blotted on Hybond P membranes (Amersham).
Immunoblotting analyses were performed using either the anti-Myc antibody 9E10
(Roche Diagnostics) or an anti-pan cadherin antibody (Sigma) as a loading
control.

Electron microscopy

Wild-type and mutant larvae were fixed with 2.5% glutaraldehyde in PBS for
30 minutes each at ambient temperature and then on ice. After washing with
PBS, the larvae were postfixed with 1% osmium tetroxide in 100 mM phosphate
buffer pH 7.2 for 1 hour on ice, washed with H20, stained with 1%
aqueous uranylacetate for 1 hour, dehydrated in a graded series of ethanol and
finally embedded in Epon. Ultrathin sections were stained with uranyl acetate
and lead citrate and viewed in Philips CM10 electron microscope. In parallel,
Toluidine Blue stained Epon sections of 0.5 μm of the same specimen were
prepared for light microscopy.

Cell transplantations, bead implantations and SU treatments

Donor embryos were injected at the one- to four-cell stage with
biotin-dextran (Fig. 5A-C) or
rhodamin-dextran (Fig. 5D-F)
(Molecular Probes), and transplanted at the shield stage into recipient
embryos from a cross of two pia heterozygous parents. Alternatively,
h2a::h2a-GFP transgenics were used as donors
(Fig. 5G-I)
(Pauls et al., 2001). Chimeric
embryos were analyzed either by in situ hybridization against pit1 or
pomc, followed by anti-biotin staining with the Vectastain Elite ABC
kit (Vector Laboratories), or by Acridine Orange or anti-Prl immunostaining,
followed by fluorescent evaluation in two different channels. Genotyping of
recipients was performed with genomic DNA extracted from clipped tails lacking
wild-type donor cells, as described above. Implantations of Fgf3-loaded beads
into embryos of piat25215 or liat24149
intercrosses were carried out as described
(Herzog et al., 2004a).
Embryos were genotyped after photography as described above (pia) or
by Herzog et al. (Herzog et al.,
2004a) (lia). Treatments with the Fgfr inhibitor SU5402
(Calbiochem) were carried out as previously described
(Herzog et al., 2004a).

pia encodes the achaete-scute homologue Ascl1a.
(A) (Top) Genetic map of a region of linkage group 4 (LG4), showing
positions of the piat25215 mutation, the ascl1a
gene and some markers used for mapping. Genetic distances of markers from the
top of LG4 according to
(http://zfin.org/cgi-bin/mapper_select.cgi)
are indicated. (Bottom) Schematic representation of Ascl1a protein: the basic
helix-loop-helix domain (blue and green) and the piat25215
mutation (red) are indicated. (B) Anti-Myc western blot, showing that
translation of chimeric ascl1a-myc mRNA is efficiently
blocked in the presence of ascl1a MO (upper panel). The same blot was
probed with an anti-pan-cadherin antibody for loading control (lower panel).
(C-H) Whole-mount in situ hybridization detecting expression of
prl (26 hpf; frontal view, dorsal up; C-E) and pomc (48 hpf;
dorsal view, anterior towards the left; F-H) in wild-type (wt; C,F),
piat25215 mutant (pia; D,G) and ascl1a
morphant embryos (ascl1aMO; E,H). (F-H) pomc-positive cells
of the adenohypophysis are indicated with arrowheads, pomc-positive
cells of the arcuate nucleus in the hypothalamus are indicated with arrows.
(I-K) In situ hybridization at 48 hpf for the pituitary marker
prl (p) and the epiphysis marker opsin (o). Injected BAC DNA
usually does not distribute uniformly, but leads to chimeric embryos with only
a subset of cells containing the injected DNA. Accordingly, the rescued
pia embryo in K displays strong prl expression (arrow), but
still lacks opsin expression in epiphysis, another phenotypic trait
caused by loss of Ascl1a function (Cau and
Wilson, 2003). The genotype of the embryo was further confirmed
via PCR (see Materials and methods). (L-N) elavl3 in situ
hybridization at 11 hpf, dorsal views, anterior towards the left,
demonstrating that wild-type Ascl1a fused to the VP16 transactivation domain
can induce primary neurons in inter-proneural domains of the neural plate (M),
whereas a fusion between VP16 and the truncated Ascl1a protein encoded by the
piat25215 allele is ineffective (N). hpf, hours post
fertilization.

RESULTS

The pia locus encodes ascl1a

During a large-scale screen for zebrafish mutants with altered growth
hormone (gh) expression, we had isolated one ENU-induced
pituitary-absent (pia) mutation
(Herzog et al., 2004b).
Segregation linkage analysis revealed that pia maps to a defined
interval on linkage group 4 (Fig.
1A, upper panel; see Materials and methods). One of the genes that
had been previously mapped to this genomic location (see
http://zfin.org/cgi-bin/mapper_select.cgi)
was the zebrafish achaete-scute homologue ascl1a,
which appeared to be a reasonable candidate, as it is expressed in the
pituitary (Allende and Weinberg,
1994; Wullimann and Mueller,
2002). Sequencing of the ascl1a gene from genomic DNA of
pia mutant embryos uncovered a C to A mutation at coding nucleotide
210, changing a tyrosine residue to a premature stop codon. This results in a
truncated Ascl1a protein that lacks all amino acid residues after position 69,
including the entire basic DNA-binding domain and the HLH domain
(Fig. 1A, lower panel). The
found ascl1 mutation generates an RFLP (see Materials and methods),
which we used to genotype additional pia homozygotes, revealing no
recombination between ascl1 and pia in 215 tested embryos
(430 meioses; <0.2 cM).

To further confirm that the defects observed in pia are due to a
loss of Ascl1a function, we phenocopied the pia defects in wild-type
embryos by knocking down ascl1a with antisense morpholino
oligonucleotides (MO) (Nasevicius and
Ekker, 2000). The used MO targeting a sequence covering the
ascl1a start codon efficiently blocked ascl1a translation,
as revealed by anti-Myc western blot analysis of protein extracts from embryos
injected with mRNA encoding an Ascl1a-Myc fusion protein
(Fig. 1B). Injection of the
acsl1a MO into wild-type embryos led to a complete loss of
adenohypophyseal prolactin (prl, 93% affected,
n=58) and pomc (86% affected, n=48) expression, a
phenotype indistinguishable from that of pia mutants
(Fig. 1C-H). Furthermore, we
were able to rescue prl and pomc expression in pia
mutants upon injection of BAC DNA containing the ascl1a gene
(Fig. 1I-K; n=7; and
data not shown).

Finally, to investigate the severity of the piat25215
mutation, we studied the effect of ascl1a overexpression on
neurogenesis in the neural plate of zebrafish embryos. Although injection of
mRNA encoding wild-type ascl1a had no effect (data not shown),
injection of mRNA encoding a Ascl1a-VP16 fusion protein (0.6 ng/μl) led to
a massive increase in the number of elavl3-positive neural precursor
cells (Fig. 1M; 78/78) (compare
with Bae et al., 2005;
Kim et al., 1997). By
contrast, the corresponding fusion between the VP16 transactivating domain and
the truncated part of Ascl1a encoded by the piat25215
allele was completely ineffective, even when injected at 60-fold higher
molarity (12 ng/μl) (Fig.
1N; 0/42). In summary, these data indicate that the pituitary
phenotype of pia mutants is caused by a null mutation in the
zebrafish achaete scute homologue acsl1a.

ascl1a is expressed in the adenohypophysis and in adjacent
domains of the diencephalon

To gain more detailed information about the spatiotemporal dynamics of
adenohypophyseal ascl1a expression, we performed whole-mount in situ
hybridization from early segmentation stages to 120 hpf. First expression of
ascl1a in the pituitary placode could be detected at the 20-somite
stage (18 hpf; data not shown), coincident with the onset of expression of
other pituitary regulators such as lim3 (lhx3 - Zebrafish
Information Network), pitx3 and pit1. At 22 hpf and 24 hpf,
ascl1a was co-expressed with pitx3 in all adenohypophyseal
precursor cells at the anterior neural ridge
(Fig. 2A-D), a subset of which
also displayed expression of prl
(Fig. 2E,F). Consistently, at
26 hpf, ascl1a transcripts were uniformly distributed throughout the
entire adenohypophyseal anlage, including anterior-most cells, which give rise
to lactotropes and corticotropes, and more lateral-posterior cells, which most
probably form somatotropes and thyrotropes
(Fig. 2G) (cf.
Herzog et al., 2004a;
Nica et al., 2004). This
uniform distribution of ascl1a transcripts persisted until 72 hpf
(see Fig. 2H for 40 hpf). Then,
expression became confined to anterior and posterior regions of the
adenohypophysis, displaying a pattern resembling that of pomc
expression (Herzog et al.,
2004a), whereas the medial domain, characterized by the expression
of gh and tsh (Herzog et
al., 2004a), became devoid of ascl1a expression
(Fig. 2I,J). This spatial shift
from a uniform to a restricted distribution of transcripts suggests that
ascl1a might initially be required in all pituitary cells, while
later playing more restricted roles in particular cell types only. At all
investigated stages (24 hpf -120 hpf), ascl1a in addition to the
adenohypopyseal primodium itself was also expressed in adjacent cells of the
diencephalon (Fig. 2C-J), such
as the posterior ventral hypothalamus (pvh;
Fig. 2I,J).

Most probably owing to the additional round of genome duplication that
occurred during teleost evolution
(Postlethwait et al., 1998),
the zebrafish has a second ascl1 gene, named ascl1b
(Allende and Weinberg, 1994).
To study whether partial redundancy between Ascl1a and its paralog Ascl1b
might account for the slightly weaker phenotype of pia mutants
compared with fgf3 mutants, we also analyzed the expression of
ascl1b (Allende and Weinberg,
1994). However, at all stages examined (24-120 hpf),
ascl1b expression was excluded from the adenohypophysis
(Fig. 2K,L; and data not
shown), indicating that the regulation of adenohypophysis development has been
dedicated to the ascl1a paralog only.

ascl1a/pia is required for proper initiation of pituitary
specification and terminal differentiation of all adenohypophyseal cell
types

Thus far, the pituitary defects of pia mutants had been analyzed
only briefly, revealing the absence of gh, pomc, prl and
thyroid-stimulating hormone (tsh) transcripts at late larval
stages (120 hpf) (Herzog et al.,
2004b). We extended such expression analyses, looking at
additional markers and at earlier stages of development. The placodal
ectoderm, from which the zebrafish pituitary originates, specifies from late
gastrulation through mid segmentation stages, and is set up normally in
pia mutants, as revealed by in situ hybridization against
eya1 transcripts (data not shown) (cf.
Herzog et al., 2004a).
Specification of the pituitary itself starts around 19 hpf, as indicated by
expression initiation of adenohypophysis-specific marker genes, such as the
pan-pituitary marker lim3
(Glasgow et al., 1997). At 25
hpf, lim3 expression was present in pia mutants, but the
staining was very diffuse and strongly reduced compared with wild-type
siblings (Fig. 3A). More
moderately reduced expression was also observed for pitx3
(Dutta et al., 2005;
Zilinski et al., 2005),
another marker of the entire adenohypophyseal anlage
(Fig. 3C), and for
ascl1a itself (Fig.
3E). Interestingly, expression levels of all three genes appeared
to recover during later stages of development, when the pituitary of
pia mutants was of reduced size (see also below); however, lim3,
pitx3 and ascl1a hybridization signals were of normal intensity
(Fig. 3B,D,E, inset).

ascl1a is expressed in the adenohypophysis and the adjacent
diencephalon. All panels show double (A-F) or single (G-L)
in situ hybridization with probes indicated in the lower right-hand corners
and ages of specimen in the upper right-hand corners. (B,D,F) Same embryos as
in (A,C,E), after the red staining has been washed out. (A-F) Dorsal views,
anterior towards the left; (G-L) lateral views, anterior towards the left,
dorsal upwards. Arrows indicate adenohypophysis (G-L) or its anlage at the
anterior neural ridge (A-F). ah, adenohypophysis; nh, neurohypophysis; pvh,
posterior-ventral hypothalamus.

By contrast, expression of other regulator genes was completely lacking in
pia mutants from earliest stages onwards. Thus, neurod,
another bHLH gene that like ascl1a is expressed throughout the
adenohypophysis of wild-type embryos
(Blader et al., 1997;
Mueller and Wullimann, 2002),
was absent in the pituitary of pia mutants at all investigated stages
(Fig. 3F; and data not shown).
Similarly, expression of pit1, which encodes a POU domain
transcription factor required for transcriptional activation of prl,
gh and tsh, and for repression of pomc
(Nica et al., 2004), was
absent in pia mutants at 20 hpf, shortly after its expression
initiation in wild-type embryos (data not shown) and at all investigated later
stages (see Fig. 3G,H for 25
hpf and 72 hpf).

Early defects in pituitary formation were also visible at the morphological
level. Using Nomarski optics, the adenohypophysis can normally be recognized
as a separate organ from 25 hpf onwards. However, in pia mutants,
adenohypophyseal borders appeared much less distinct than in their wild-type
sibling embryos (Fig. 4A,B; 26
hpf). Together with the decreased intensity of early marker gene expression,
this suggests that ascl1a is crucial for the proper onset of
adenohypophysis specification and organ formation.

We also studied the expression of the different hormone genes at early time
points of pituitary development. In zebrafish, terminal differentiation of the
different adenohypophyseal cell types occurs in sequential steps between 22
hpf and 48 hpf (Herzog et al.,
2003). The first cells that undergo terminal differentiation are
the lactotropes, indicated by the expression of prolactin
(prl), which is initiated at 22 hpf. The next lineages to
differentiate are the corticotropes and melanotropes, which express
pomc from 24 hpf onwards (Herzog
et al., 2003). The somatotropes, marked by expression of
gh (Herzog et al.,
2003), the thyrotropes, marked by co-expression of tsh
and gsu (Herzog et al.,
2003; Nica et al.,
2004), and the gonadotropes, marked by expression of gsu
(Nica et al., 2004), start to
differentiate between 36 and 42 hpf. In pia mutants, no prl
transcripts could be detected at 26 hpf
(Fig. 1C,D), and no gh
or tsh transcripts at 48 hpf (Fig.
3I,K). In addition, transcripts of pomc and gsu
were absent from pia mutant pituitaries at 26 hpf (data not shown)
and at 48 hpf (Fig. 1F,G;
Fig. 3J). In summary, this
indicates that Ascl1a is required for the initiation of cognate hormone genes
of all adenohypophyseal cell types.

pia mutants display indistinct early pituitary morphology,
followed by a transient phase of adenohypophyseal cell death and the formation
of a smaller, but distinct, pituitary gland with cells of rather primitive
ultrastructure. (A-L) Nomarski images of live pia
mutant embryos (pia) and their corresponding wild-type siblings (wt).
(F,H,I-L) Images are superimposed by Acridine Orange staining for apoptosis.
(A,B,E-J) Frontal views, dorsal upwards; (C,D) lateral views, anterior towards
the left, dorsal upwards; (K,L) ventral views, anterior towards the right.
Ages of embryos are indicated in upper right-hand corners of wild types.
Genotypes were determined via PCR after photography. Arrowheads in A-J
indicate borders of the pituitary gland; in K,L, pituitary borders are
outlined by dots. (M-T) Pituitary ultrastructure at 120 hpf;
longitudinal sections, anterior towards the left, dorsal towards the top.
(M,O) Toluidine Blue staining; (N,P-T) electron micrographs. (M-P) The border
of the pituitary is indicated by arrows; (N,P) the border between
adenohypophysis (ah) and neurohypohysis (nh) is outlined by black dots. (Q-T)
Higher magnifications of regions indicated by red arrows in N,P. Vesicles (as
in T, indicated by arrows) were seen in three out of ∼20 adenohypophyseal
cells present in the section of the pia pituitary (P). They could
contain matrix proteins and hormone-binding proteins, which can be made even
in the absence of hormone production (compare with
Norris, 1997). Scale bars: 30μ
m in M-P; 1 μm in Q-T.

In a previous study, we have shown that in fgf3 mutant zebrafish
embryos, failed pituitary specification is followed by destruction of the
entire organ, driven by apoptosis of non-differentiated adenohypophyseal cells
between 28 and 32 hpf (Herzog et al.,
2004a). As early adenohypophyseal specification seems similarly
disrupted in pia mutants, we determined apoptosis rates in
pia mutant pituitaries, performing Acridine Orange staining at
various stages of development. At 24 hpf, no Acridine Orange-positive cells
could be detected at the anterior neural ridge, where the adenohypophyseal
primordium is located (data not shown; two genotyped mutants). However, at 27
hpf (data not shown; four genotyped mutants) and more dramatically at 30 hpf
(Fig. 3E-H; three genotyped
mutants), mutant embryos displayed significantly increased numbers of
apoptotic cells within the adenohypophyseal anlage. At this stage, the mutant
organ was morphologically much more distinct than at 26 hpf (compare
Fig. 4G with 4B), although
smaller and flatter compared with the pituitary of 30 hpf wild-type siblings
(compare Fig. 4G with 4E). In
addition to the adenohypophysis itself, Acridine Orange-positive signals were
found in regions adjacent to the organ
(Fig. 4H). These signals most
probably represent debris of apoptotic cells that have been extruded from the
adenohypophysis, as previously shown in fgf3 mutants by cell-tracing
experiments (Herzog et al.,
2004a). At 36 hpf, the number of dying cells in pia
mutants was significantly decreased compared with the situation at 30 hpf
(three genotyped mutants; Fig.
4I,J), while no apoptotic adenohypophyseal cells at all could be
detected at 48 hpf (data not shown; three genotyped mutants), and at 52 hpf
(two genotyped mutants; Fig.
4K,L). In contrast to fgf3 mutants
(Herzog et al., 2004a),
however, pia mutants of these late stages had a morphologically
distinct adenohypophysis of smaller size
(Fig. 4C,D), indicating that a
subset of non-differentiated adenohypophyseal cells must have survived the
transient phase of cell death. Together, these results indicate that in
addition to, or as a consequence of, its role during cell differentiation
Ascl1a is also required for the survival of most, but not all adenohypophyseal
cells.

As mentioned above, surviving cells in the pituitary rudiment of later
stage pia mutants displayed rather normal levels of lim3,
pitx3 and ascl1a transcripts
(Fig. 3B,D,E). In addition,
they lacked expression of genes marking other derivatives of the placodal
ectoderm such as crystallin bb1 (lens tissue;
Fig. 3L) or omp
(olfactory epithelium; Fig.
3M), ruling out trans-differentiation to these other placodal cell
types, and strongly suggesting that pituitary identity was maintained to some
extent. To gain further insight into the nature of cells in the pituitary
rudiment, we carried out transmission electron microscopy. At 120 hpf, the
pituitary of pia mutants showed a neurohypophyseal compartment of
rather normal size and morphology (compare
Fig. 4M,N for wild type with
Fig. 4O,P for pia).
However, striking differences were apparent in the adenohypophysis. In
wild-type siblings, most, if not all, adenohypophyseal cells contained
secretory vesicles. By morphology, at least two types of secretory vesicles
could be distinguished (Fig.
4Q,R), most probably reflecting different hormone-producing cell
types (Nica et al., 2006). By
contrast, the adenohypophysis of pia mutants not only contained fewer
cells (Fig. 4O,P); ∼90% of
the cells also displayed a rather primitive ultrastructure. Thus, their
cytoplasm lacked an elaborated endoplasmic reticulum and secretory vesicles,
whereas the chromatin of the nucleus was very homogeneous
(Fig. 4S). However, a few
adenohypohyseal cells of pia mutants did contain a particular type of
secretory vesicles (Fig. 4T),
although in smaller numbers than in wild-type siblings, and despite the
absence of hormone synthesis. This suggests that pia mutant
adenohypophyseal cells in their entirety are not fully differentiated,
although they differ in their exact specification state.

Ascl1a is required cell-autonomously in adenohypophyseal cells

As described above, ascl1a is expressed not only in
adenohypophysis itself but also in adjacent tissues, such as the ventral
diencephalon, the source of signaling proteins such as Shh or Fgf3, both of
which are required for proper pituitary development
(Herzog et al., 2004a;
Herzog et al., 2003;
Sbrogna et al., 2003). Thus,
Ascl1a might either have an essential intrinsic role in adenohypophyseal cells
themselves, or might influence adenophypophysis indirectly via the
diencephalon. In order to distinguish between these possibilities, we
generated chimeric embryos. For chimeras with donor cells in the pituitary,
labeled cells were transplanted at early gastrula stages into regions of host
embryos ventral to the animal pole, whereas for telencephalic or diencephalic
clones, cells were transplanted right at the animal pole, or slightly animal
of the dorsal shield, respectively (compare with
Herzog et al., 2004a).
Chimeric embryos were raised to 26-48 hpf, and stained either for
pit1 transcripts to evaluate early adenohypophyseal specification,
with Acridine Orange to evaluate cell survival, or for Prl protein to evaluate
terminal endocrine differentiation. When wild-type cells ended up in the
telencephalon, which is located adjacent to the adenohypophyseal anlage early,
or in the ventral diencephalon, which is in close proximity to the
adenohypophysis later (Herzog et al.,
2004a), no induction of adenohypophyseal pit1 expression
was obtained (16/16 embryos; Fig.
5B). By contrast, when wild-type donor cells were located within
the adenohypophysis of 26 hpf pia mutant hosts, most of them were
pit1 positive (4/5 embryos; Fig.
5C; pit1-negative wild-type cells most probably represent
corticotropes, melanotropes or gonadotropes). Similarly, at 32 hpf,
transplanted wild-type cells within the adenohypophysis of pia mutant
hosts all were Acridine Orange negative, while many of the host pituitary
cells were apoptotic (nine embryos; Fig.
5D,E). In reverse, some of the ascl1a morphant cells
transplanted into wild-type pituitaries became apoptotic, while the host
tissue was completely Acridine Orange negative (11 embryos;
Fig. 5F). Finally, many of the
wild-type cells transplanted into pia mutant pituitaries contained
pomc transcripts (four embryos; data not shown) or Prl protein at 48
hpf (eight embryos; Fig. 5I),
while pia mutant cells were Pomc- and Prl-negative
(Fig. 5H,I; and data not
shown). In summary, this strongly suggests that Ascl1a is required in a
cell-autonomous fashion in the adenophypophysis itself to promote early steps
of specification, cell survival and terminal endocrine differentiation. By
contrast, ascl1a expression in telencephalon and diencephalon appears
to be dispensable for proper adenohypophysis development, consistent with the
normal expression of all investigated marker genes in the forebrain of mutant
embryos (nkx2.1a, Fig.
3N; fgf3, Fig.
6C,D; shh, data not shown).

The findings that fgf3 and ascl1a mutants display similar
pituitary phenotypes, and that Fgf3 acts as an extrinsic signal
(Herzog et al., 2004a) and
Ascl1a as an intrinsic transcription factor, suggests that Ascl1a might act
downstream of Fgf3 signaling to mediate at least some of its effects. This
notion is further supported by the lack of adenohypophyseal ascl1a
expression in fgf3/lia mutants at 28 hpf
(Fig. 6A,B), before the
appearance of massive cell death (Herzog
et al., 2004a), although in reverse, diencephalic fgf3
expression is normal in ascl1a/pia mutants
(Fig. 6C,D). Similarly,
adenohypophyseal ascl1a expression was absent in 28 hpf wild-type
embryos after treatment with the Fgf receptor inhibitor SU5402 from 18-22 hpf
(data not shown). To investigate more directly whether Ascl1a is required to
mediate diencephalic Fgf3 signaling, we implanted Fgf3-loaded beads into the
diencephalon of embryos obtained from pia+/- intercrosses
at 18 hpf, briefly before first adenohypophyseal defects in pia
mutants become visible. Such beads caused a significant normalization of
adenohypophyseal ascl1a expression in pia mutants at 28 hpf
(4/6; Fig. 6F). However, they
failed to rescue pit1 expression in pia mutants of the same
age (5/5; Fig. 6I), in contrast
to the striking rescue obtained in control implantations into
fgf3/lia fish (6/6; Fig.
6J). This indicates that in the absence of Ascl1a, Fgf3 signaling
can activate the expression of ascl1a itself, but not the expression
of downstream genes, consistent with the notion that Fgf3 requires Ascl1a to
mediate at least some of its effects on the adenohypophysis.

DISCUSSION

Zebrafish Ascl1a is required for endocrine differentiation in the
adenohypophysis

Here, we extend this list of Ascl-dependent cell types, showing that
zebrafish Ascl1 is required for endocrine differentiation of adenohypophyseal
cells. The adenohypophysis derives from the placodal ectoderm, in close
proximity to the olfactory epithelium
(Dubois and ElAmraoui, 1995).
In mouse, the ascl1a homologue Mash1 is required for early
olfactory development (Guillemot et al.,
1993), while in zebrafish ascl1a mutants, the olfactory
epithelium develops normally, indicated by unaffected expression of
omp (Fig. 3). This is
most probably due to the presence of the ascl1a paralog
ascl1b in this tissue (Fig.
2) (Allende and Weinberg,
1994). Alternatively, it might be due to functional redundancy
between Ascl1a and other proneural bHLH proteins, similar to the situation
described for zebrafish Ascl1a and neurogenin 1 during epiphysial neurogenesis
(Cau and Wilson, 2003).

It is unclear why, in vertebrates, Ascl proteins in addition to neurons are
particularly required for the specification of endocrine fates. In the case of
the adenohypophysis and the olfactory epithelium, it might result from their
common developmental and evolutionary origin. Thus, both tissues derive from
the placodal ectoderm, and according to a current model, they have evolved
from a common chemoreceptive structure already present in Bilaterian ancestors
(De Velasco et al., 2004;
Gorbman, 1995).

Different roles of Ascl1a during zebrafish adenohypophysis
development

In line with the proposed common origin of adenohypophysis and neural
structures, we found striking similarities in the modes of Ascl1 action during
endocrine differentiation in the zebrafish adenohypophysis and neurogenesis in
Drosophila and mouse. In Drosophila, the function of
asc members is divided into distinct developmental steps
(Brunet and Ghysen, 1999;
Jan and Jan, 1994;
Westerman et al., 2003). The
first is the establishment of neural precursors out of a population of
progenitors (proneural function), the second is the specification of specific
neural fates (subtype specification step). The same is true for Mash1 during
neural development in the mouse. Thus, in the subventricular zone of the
ventral telencephalon, Mash1 is required for the formation of a pool of neural
precursor cells (Casarosa et al.,
1999), whereas in autonomic ganglia, the olfactory epithelium and
some regions of the brain, it activates the expression of specific neuronal
differentiation genes and initiates neuronal differentiation
(Cau et al., 1997;
Guillemot et al., 1993;
Hirsch et al., 1998;
Parras et al., 2002).

A similar biphasic proneural-like and subtype-specification function also
appears to apply for Ascl1a in the zebrafish adenohypophysis. Thus, in the
absence of Ascl1a function, all adenohypophyseal cell types fail to
differentiate, in line with a general role of Ascl1a during the specification
of pituitary precursor cells. Actually, Ascl1a is the first transcription
factor shown to be required for the proper differentiation of all
adenohypophyseal cell types, whereas mutants in all other previously described
transcription factors only lack particular adenohypophyseal lineages (for a
review, see Zhu and Rosenfeld,
2004).

In somatotropes and thyrotropes, ascl1a is switched off after
endocrine differentiation and the initiation of cognate hormone gene
expression (Fig. 2). This
indicates that, in these cell types, ascl1a is exclusively involved
in earlier specification steps, such as the activation of pit1, the
expression of which fails to be initiated in ascl1a mutants
(Fig. 3). By contrast,
ascl1a expression persists in the Pomc lineage
(Fig. 2), suggesting that in
these cell types, Ascl1a might be involved in later, subtype-specific
differentiation processes. Interestingly, the zebrafish pomc promoter
contains E-boxes, consensus binding sites for bHLH transcription factors
(Liu et al., 2003), suggesting
that Ascl1a might be involved in the transcriptional activation of the
pomc gene. In summary, it appears that Ascl1a regulates
adenohypophysis development at multiple levels, acting during both early and
terminal steps of the transcriptional control cascade.

Differential apoptosis within the ascl1a mutant pituitary
and the nature of surviving cells

In addition to cell specification processes, ascl1a is required
for the survival of some, but not all adenohypophyseal cells, allowing us to
distinguish two cell populations: one in which cell differentiation and cell
survival are strictly coupled; and another in which cells can survive in an
undifferentiated state (Fig.
4). In mutant embryos, cell death occurs during a rather narrow
time window (2-3 hours), several hours after the failed differentiation of
lactotropes and corticotropes, but before somatotropes and thyrotropes would
differentiate under wild-type conditions
(Herzog et al., 2003). These
surviving cells of ascl1a-deficient pituitaries have adenohypophyseal
character, as revealed by the presence of lim3, pitx3 and
ascl1a transcripts at 72 hpf (Fig.
3), and their ultrastructural morphology at 120 hpf, which
resembles that of undifferentiated adenohypophyseal precursor cells
(Fig. 4). In the mouse
olfactory epithelium, where Mash1 plays a subtype-specific role to drive final
steps of neural differentiation (see above), loss of Mash1 leads to massive
cell death; however, apoptosis only starts after the initiation of the
differentiation arrest (Cau et al.,
1997). Assuming that the same is true for the zebrafish
adenohypophysis, this would mean that apoptosis (between 30 and 32 hpf) occurs
only in the early differentiating lineages (corticotropes, melanotropes and
lactotropes; 24-28 hpf). Along the same lines, surviving cells would represent
somatotrope and thyrotrope precursors, which normally differentiate later
(42-48 hpf). However, their differentiation in the pituitary rudiment of
ascl1a mutants would be blocked because of the absence of Pit1
(Fig. 3), which is absolutely
required for tsh and gh hormone gene expression
(Nica et al., 2004). Clearly,
this is only one of several possible interpretations, and further experiments,
such as transgene-driven re-introduction of pit1 gene products into
the pituitary rudiment of pia mutants will be necessary to prove this
notion. In addition, it appears that even within this population of surviving
cells, Ascl1a might have differential effects, as suggested by the different
ultrastructure of adenohypophyseal cells in older mutants
(Fig. 4).

Does Ascl1a act downstream of Fgf3 and upstream of Neurod?

In mouse, out of all bHLH proteins, only Neurod1 has been described in the
context of pituitary development. In particular, Neurod1 has been shown to
bind and activate the Pomc promoter, acting in concert with a Pitx
homeodomain protein and another bHLH factor
(Horton et al., 1999;
Poulin et al., 2000;
Poulin et al., 1997;
Westerman et al., 2003). To
search for bHLH proteins that might act in parallel or downstream of Ascl1a,
we studied the expression pattern of other zebrafish achaete scute
and atonal homologues. However, we only found neurod
(Fig. 3) to be expressed in the
adenohypophysis, whereas ascl1b, neurod2, ngn1, atho1.1, atho1.2,
atho2a/ndr1a, atho2b/ndr1b, ath3/neurod4,
atho4/ngn3 and ath5 were not (H.M.P. and M.H., unpublished).
Interestingly, in ascl1a mutants, neurod expression was
absent at all investigated stages, suggesting that it acts downstream of
Ascl1a. However, in contrast to Neurod1-deficient mice, which show
moderate defects during corticotroph differentiation
(Lamolet et al., 2004), we
failed to observe any abnormalities during zebrafish pituitary development
when knocking down zebrafish Neurod with specific antisense morpholino
oligonucleotides (H.M.P. and M.H., unpublished). This indicates that Neurod is
neither an essential partner, nor an essential downstream mediator of Ascl1a
during zebrafish pituitary development. To address whether Neurod might
nevertheless mediate Ascl1a function, e.g. in redundancy with another as yet
unidentified factor, it will be necessary to generate transgenic lines to
drive Ascl1a-independent adenohypophyseal neurod expression,
investigating whether forced neurod expression is sufficient to
rescue the adenohypophyseal defects of ascl1a mutants.

A similar transgenic approach will be necessary to provide ultimate proof
for a role of Ascl1a downstream of Fgf3 signaling from the ventral
diencephalon. For several reasons, such a role seems quite likely. First,
fgf3 mutants show a similar combination of failed specification and
apoptosis of adenohypophyseal cells
(Herzog et al., 2004a), as
described here for ascl1a mutants. Second, fgf3 mutants lack
adenohypophyseal ascl1a expression, and third, implantation of
Fgf3-loaded beads into ascl1a mutants can enhance adenohypophyseal
ascl1a expression, while genes downstream of Ascl1a such as
pit1 do not respond. Clearly, without the aforementioned rescue of
fgf3 mutants by forced ascl1a expression, these data do not
rule out that Ascl1a acts in parallel to, rather than downstream of, Fgf3. In
any case, Fgf3 must have other mediators in addition to Ascl1a, given that the
pituitary in fgf3 mutants is more severely affected than in
ascl1a mutants, with apoptosis of all rather than a subset of
adenohypophyseal cells.

De Velasco, B., Shen, J., Go, S. and Hartenstein, V.
(2004). Embryonic development of the Drosophila corpus cardiacum,
a neuroendocrine gland with similarity to the vertebrate pituitary, is
controlled by sine oculis and glass. Dev. Biol.274,280
-294.

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